Summary
The quality control assessment of lactic acid bacteria (LAB) cultures has been confirmed as an effective way to enhance the viability and functionality of LAB strains for fermentation procedures. To buttress this assertion, we developed a protocol that elucidates how LAB cultures are activated and cultivated for fermentation and bioprocessing procedures.
Abstract
Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as yogurt and cheese. LAB predominantly produce lactic acid as a major end product of fermentation, and they synthesize important metabolites that impart the organoleptic characteristics of fermented food products. LAB are fastidious bacteria that thrive in many environments when adequate nutritional requirements are fulfilled. The demand for superior LAB dairy starter cultures for fermentation applications in the food and dairy industry, has resulted in the need to provide viable and active cultures for all bioprocessing operations. The development of a standard protocol for ensuring the viability and enhanced functionality of LAB cultures in the laboratory as well as dairy processing environments is thus very critical. In addressing concerns linked to resuscitating weak, stressed, and injured LAB culture cells, a protocol that vividly outlines salient steps to recover, enhance cell regeneration, and improve metabolic functionality of LAB strains is of the utmost importance. The maintenance of culture purity, functionality, and viability for LAB starter cultures is likewise critical. Therefore, adherence to a unique protocol guideline will result in the promotion of fermentation performance for many LAB strains dedicated to fermentation and biotechnology processes. As a result, the Food Microbiology and Biotechnology Laboratory at North Carolina Agriculture and Technical State University has developed a standard protocol for the activation and quality control of selected LAB strains that has resulted in highly functional and viable LAB culture strains employed for fermentation research. The adaptation and recommendation of a protocol such as this for use in the dairy and food industry will help to ensure LAB viability and functionality for many applications.
Introduction
Lactic acid bacteria (LAB) are a group of uniquely diverse bacteria that have industrial potential. Strains belonging to Lactobacillus delbreuckii subsp. bulgaricus and Streptococcus thermophilus are mostly used as dairy starter cultures for fermented dairy food products such as yogurt1. Selected LAB strains are also classified as probiotics as they confer health benefits to humans when dosages are adequately administered2. Lactic acid bacteria are also gram-positive, non-spore-forming, non-respiring but aerotolerant microorganisms that are generally characterized by the production of lactic acid as a key fermentation product. LAB also synthesizes essential metabolites, for example, organic acids, bacteriocins, and other antimicrobial compounds3 that can inhibit a broad spectrum of foodborne pathogens4. Lactic acid, a major end product of carbohydrate catabolism and a by-product of LAB fermentation, is an organic metabolite that possesses antimicrobial properties and is potentially useful for food biopreservation applications3,5,6. Furthermore, the organic acids produced by LAB impart the flavor, texture, and aroma of foods, thus consequently enhancing their overall organoleptic properties5,6. The distinct nutritional requirements of LAB coupled with their ubiquitous nature, ultimately enable the bacteria to easily thrive in different environments such as dairy-based foods, fermented foods, vegetables as well as in the human gut7.
There is a growing demand for starter cultures from LAB for yogurt production and many diverse dairy applications8,9, hence critical attention and established scientific techniques should be adhered to, in LAB strains cultivation, as well as in the activation of both lyophilized and isolated strains as this activity is vital for enhanced fermentation performance. The Food Microbiology and Biotechnology laboratory, therefore, actively engages in suitable technology development geared toward the activation, superior growth, and fermentation characteristic of LAB strains isolated from fermented dairy products as well as from industrial starter cultures employed for yogurt production. Furthermore, it is noteworthy that LAB culture strains industrially produced undergo preservative activities such as freeze-drying and frozen storage, causing cell stress and injury, as a result of the cold shock process they are subjected to10. In limiting, the viability challenges and improving the functionality of LAB strains obtained from either isolated food products or freeze-dried products, it is important to properly activate these cultures as a form of quality control to enhance their fermentative characteristic8. In this study, the objective was to develop an in-house quality control protocol for the activation and growth of L. delbrueckii subsp. bulgaricus culture strains that ultimately promoted viable LAB growth, as well as enhanced the fermentation performance and the metabolic functionality of LAB strains. This protocol could ultimately be adapted (using optimal growth media and appropriate culturing conditions) for the cultivation of other LAB strains for fermentation research, as well as for industrial purposes or bioprocessing operations. This LAB activation and quality control protocol will therefore ensure superior viable dairy starter cultures are obtained and potentially functional for diverse applications in the global dairy and food industry.
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Protocol
1. General materials and methods
- Source of Lactobacillus delbrueckii subsp. bulgaricus
- Obtain L. bulgaricus strains from reliable sources.
NOTE: In this study, a total of five (5) L. bulgaricus strains were used in the quality control study (Table 1). Two strains of freeze-dried L. bulgaricus for the industrial production of fermented milk products were provided by Dr. Albert Krastanov, Department of Biotechnology at the University of Food Technologies, Plovdiv, Bulgaria. Two strains isolated from commercial yogurt products available in the US market were obtained from the -80 °C stock of the Food Microbiology and Biotechnology laboratory at North Carolina A&T State University and one freeze-dried strain was supplied by a vendor C. All LAB strains were kept at -80 °C until further use. Another bacterial strain (Limosilactobacillus reuteri) was obtained from the -80 °C stock of the Food Microbiology and Biotechnology laboratory at North Carolina A&T State University was also evaluated.
- Obtain L. bulgaricus strains from reliable sources.
- Standard L. MRS fermentation broth medium
- Prepare deMan, Rogosa Sharpe (MRS) medium by completely dissolving 55 g of MRS, and 0.5 g of L-Cysteine in 1 L of deionized water (DW).
- Dispense 7 mL and 2 mL of the prepared MRS solution into test tubes, respectively, autoclave at 121 °C for 15 min, and then cool at room temperature (RT).
- MRS agar medium
- Prepare MRS agar medium by completely dissolving 55 g of MRS and 0.5 g of L-Cysteine in 1 L of DW. Add agar powder (15 g), sterilize the agar medium at 121 °C for 15 min, and then cool it in a water bath.
- Pour all freshly-prepared media into sterile Petri dishes and store them at 4 °C until further needed.
- Modified reinforced clostridial medium-pyruvate (mRCM-PYR)
- Optimize a reinforced clostridial medium according to Oyeniran et al.13 and Nwamaioha and Ibrahim18, for selectivity and accurate enumeration of L. bulgaricus by dissolving 10 g of peptone #3, 10 g of beef extract, 5 g of yeast extract, 5 g of sodium chloride, 3 g of sodium acetate, 2 g of potassium phosphate dibasic, 0.1 g of uracil, 0.25 g of calcium chloride, 5 g of dextrose, 5 g of fructose, 10 g of maltose, 2 g of sodium pyruvate, 0.2% Tween 80, and 0.5 g of L- Cysteine in 1 L of DW.
- Adjust the final pH (6.0 ± 0.2) of the solution using 6 N HCl before the addition of 0.008% aniline blue and 15 g of agar. Autoclave the medium at 121 °C for 15 min, cool in a water bath, and pour into sterile Petri dishes. Store all freshly-prepared media in sterile Petri dishes at 4 °C until needed.
- Glycerol stock of LAB cultures
- Prepare glycerol stocks (50% glycerol) by diluting 100% glycerol in the same volume of DW and sterilize at 100 °C for 30 min using a dry sterilization cycle. Cool the glycerol stocks to RT and aseptically store them for further use.
- Use bacterial growth (a single colony of L. bulgaricus obtained using the developed QC protocol) from overnight cultures.
- Pipette 500 µL of the overnight cultures into 50% glycerol in a 2 mL centrifuge tube and gently mix them together. Freeze and store the glycerol stock containing the LAB cultures at an ultra-low temperature of -80 °C until needed.
NOTE: A graphical scheme of the protocol for the quality control and activation of LAB cultures is shown in Figure 1.
| No | Product Code | Sample | Source | Bacterial Composition as labeled1 |
| 1 | S9 | Pure Industrial Strain | Bulgaria | Lb. bulgaricus |
| 2 | LB6 | Pure Industrial Strain | Bulgaria | Lb. bulgaricus, |
| 3 | ATCC 11842 | Pure Industrial Strain | ATCC | Lb. bulgaricus |
| 4 | DAW | Yogurt | USA | Lb. bulgaricus, other live culture |
| 5 | E22 | Yogurt | USA | Lb. bulgaricus, other live culture |
| 6 | Reuteri | Yogurt | USA | Limosilactobacillus reuteri |
| 1Lb. =Lactobacillus | ||||
Table 1: Probiotic strains. The table lists the probiotic strains used in this study.
2. Protocol for the activation and quality control of LAB cultures
- Take the prepared glycerol stock of LAB strains (in 2 mL centrifuge tubes) from the -80 °C ultra-low freezer, and do not allow them to thaw before use.
- Clean and disinfect the opening of the centrifuge tubes with 70% alcohol, and gently vortex before use.
- Pipette about 250 µL (0.25 mL) of the stock LAB culture from the centrifuge tubes into fresh 2 mL MRS test tubes.
- Gently vortex, parafilm the test tubes, and anaerobically incubate them overnight at 42 °C for 12-16 h.
- Take about 500 µL (0.5 mL) from the overnight grown cultures from the 2 mL MRS test tubes into fresh 7 mL MRS test tubes, vortex, and anaerobically incubate them overnight at 42 °C for 12-16 h.
- Assess the microbial growth by measuring the optical density (OD) or growth of the cultures at 610 nm with a UV- visible spectrophotometer and record acceptable results between 0.7 and 0.9.
- Streak the overnight cultures from the 7 mL MRS tubes onto MRS and MRCM-PYR agar plates and incubate them anaerobically for 72 h at 42 °C.
- Pick isolated colonies from the agar plates, transfer them into fresh 7 mL MRS test tubes, gently vortex, and anaerobically incubate them overnight at 42 °C for 12-16 h.
- Store the agar plates containing the isolated strains at 4 °C in the refrigerator for a week.
- Measure and confirm the OD (between 0.7 and 0.9) from the 7 mL MRS test tubes of the LAB cultures isolated from the streaked plates at 610 nm and use them as working cultures for all related experiments.
- Perform ten-fold dilutions (serially dilute) of the grown LAB cultures from the final 7 mL MRS test tubes using 9 mL of peptone water (a physiological buffer) to obtain a 1:10 ratio.
- Finally, take about 250 µL (0.25 mL) from appropriate serial dilutions for all fermentation experiments.
- Activate the broth containing strains (250 µL) from step 2.12 by transferring them into fresh 7 mL MRS broth and incubating them anaerobically at 42 °C for 16 h.
- Continue by repeating steps 2.6-2.12 to ensure viable and superior cell growth from LAB cultures.
NOTE: All L. bulgaricus strains were activated in the freshly prepared MRS broth and were then incubated anaerobically at 42 °C for 16 h in order to reach an optical density (OD610 nm) of bacterial growth between 0.7 and 0.9. Bacterial growth was measured with a UV-visible spectrophotometer at 610 nm. The pH values of the overnight cultures were in the range of 3.5 to 5.3 as a result of the production of lactic acid, an organic end product of LAB fermentation. Safety procedures such as proper airflow circulation in the biosafety hood and avoiding burns during the use of the bunsen burner were adhered to and observed.
Figure 1: A graphical scheme of the protocol for the activation of lactic acid bacteria (LAB) cultures. The scheme provides details and the basic instruments required for the handling and activation of LAB culture strains. Please click here to view a larger version of this figure.
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Representative Results
Cell growth of the evaluated LAB strains cultivated with the quality control protocol was significantly different (P < 0.05) than the strains cultivated without this standard protocol. The QC protocol for both L. bulgaricus and L. reuteri employed a multi-subculturing approach (subculturing three times before streaking on agar plates), whereas the control procedure had subculturing done only once with all other conditions kept constant. The colony growth was also higher and well defined on the growth media plates that were cultivated using this standard QC protocol. This confirmed the effectiveness of the protocol in ensuring enhanced LAB growth through cell division in the fermentative broth, therefore, aiding enhanced cell metabolism in the LAB strains. The growth of two strains cultivated with and without the developed quality control protocol is shown in Figure 2. The strains were streaked onto agar plates after intense growth (0.7-0.9 at OD610 nm) was observed and measured. The plates were anaerobically incubated at 42 °C for 72 h after which discrete colonies were observed along the lines of streaking. Another study (Figure 3) confirmed the efficiency of the quality control protocol on the growth of Lactobacillus reuteri cultured in TPY medium and, incubated at 37 °C, whereby colonies generated by the QC protocol technique confirmed a stable steady-state growth (averagely 0.9 at OD610 nm) based on historical data. A control method of LAB activation without multiple sub-culturing that did not employ the developed QC technique resulted in an uneven growth pattern (0.9-0.75 at OD610 nm) of L. reuteri.
Figure 2: Colonial morphology of growth of L. bulgaricus strains cultivated with and without the developed quality control protocol. The strains were streaked in triplicates and were anaerobically incubated at 42 °C for 72 h. Please click here to view a larger version of this figure.
Figure 3: Behavior of L. reuteri over a period after growth in TPY medium at 37 °C using the standardized QC protocol and the conventional method of bacterial culturing (P < 0.05). The growth of L. reuteri was monitored periodically for a month and was aligned with historical data from previous years based on culture activation with the standard QC protocol. The error bars indicate the standard deviation of the OD values. Please click here to view a larger version of this figure.
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Discussion
The results of all strains evaluated with the quality control protocol and without the use of the protocol were the same, and as such, results linked to only strains (S9, and LB6) were presented. The activated LAB strains had superior cell growth that was characterized by a high intensity of cell biomass, therefore, causing a turbid appearance of the MRS fermentative broth in the test tube11. The observed cell growth after culture activation was evident between 12 h and 16 h at an anaerobic fermentative temperature of 42 °C. It was also confirmed that the LAB strains activated based on this protocol continuously had their colony morphologies transformed as a result of the multi sub-culturing procedure12 with discrete colonies visible along the lines of streaking. The control experiment did not employ this standard protocol and was only subcultured once; it, however, did not also show intense turbidity in the fermentative broth, although growth was still within the accepted range of 0.7-0.9 at OD610 nm. It was also noteworthy that streaking the control cultures on the agar plates and anaerobically incubating them under the same conditions as the standard procedure only depicted smaller and fewer colonies as compared to those observed in the standard protocol13.
This could be attributed to the weak and non-viable characteristics of the LAB cells. Consequently, the concept of sub-culturing LAB strains multiple times and streaking on agar plates, thus, results in the generation of new cells through cell division and exchange of genetic matter in the bacteria. This enhances cellular and metabolic functionality and promotes the growth of cells during fermentation operations14. In addition, stressed and injured LAB cells are also recovered based on the steady-state of growth conditions (nutritional requirements, pH, and temperature) that enable them to easily adapt and regain full cell and metabolic functionality6. Moreover, strict adherence to this protocol limits contamination of cell cultures to a greater extent, thereby ensuring only pure and superior cell colonies are isolated for fermentation purposes. Thus, repeated streaking and recovery of LAB cell cultures from agar plates provided an enhanced cell wall that was well suited for metabolic activities such as for LAB fermentation operations15. Furthermore, the study on the growth of L. reuteri based on the developed quality control protocol (Figure 3) confirms how subculturing LAB via colony to culture method yields a stable steady growth at the optimum growth range (0.9-1.0 OD610 nm) over a period as compared to subculturing directly from culture to culture (conventional method), which resulted in a sharp growth from 0.75 to 0.9 at OD610 nm. It was also evident that the QC protocol, over time, consistently yielded a stable bacteria growth pattern, which had a standard deviation (SD) of 0.1. The uneven growth of L. reuteri, however, had its SD greater than 0.1 due to the uneven growth of the bacterial cells. This uneven growth pattern, as already stated, was attributed to weak and injured cells; thus metabolic functionality of the cells decreased during the fermentation period14. The current study was also confirmed by a previous one by Ahmed et al.16 whereby, in their study, L. reuteri was propagated in trypticase peptone yeast extract (TPY) agar medium for 8 weeks at 37 °C. A contributory factor for the steady growth of L. reuteri was attributed to the method of cell cultivation and quality control technique that generated new and superiorly active cultures even after several subculturing activities during the study period.
The critical steps in this protocol include always employing new and less than a day old fermentative cultures and always having the fermentative broth or growth medium newly prepared before commencement of fermentation, and not using stored growth medium as the strength and potency of the medium decreases over an extended period of storage. In the case of using stock cultures from either a -80 °C or -20 °C, the cultures should be aseptically handled. Furthermore, if stock cultures are observed to be weak due to cold shock from the freezer, resuscitation is required as follows. Only low concentrations (about 100 µL) of the stock cultures should be inoculated in the supplemented fermentative broth (MRS) of about 2 mL for pre-anaerobic incubation for about 6 h before additional supplemented MRS of about 5-6 mL is added for final anaerobic incubation for about 16 h17. In the event of using lyophilized or freeze-dried cultures, it is highly recommended to recover the freeze-dried cultures in 3 mL of skimmed fluid milk and anaerobically incubate them for about 4-5 h before inoculating them in the supplemented fermentative broth such as MRS. These processes are key in ensuring LAB cell viability at all times13.
Another critical step is to ensure cell growth of fermentative cultures is always within the growth range of 0.7-0.9, which is usually at OD610nm. This is important as that growth range is recommended as the appropriate log or exponential phase of the bacterial growth curve17. Any optical density (OD) value above 1, is inaccurate as this confirms the fermentative cultures have reached their death phase with most dead cells and accumulation of toxic cellular waste predominantly present in the fermentative medium. In the event of reaching an OD value above 1 (OD610nm > 1)., it is recommended to dilute the cultures 2 or 3-fold or more until it reaches an OD value below 1 (OD610nm < 1)18. Another point of concern is to appropriately anaerobically incubate the streaked plates with the fermentative cultures. Although most LAB are facultative anaerobes, enhanced growth is limited in the presence of oxygen; hence the preference for anaerobic conditions is required to promote efficient growth. It is recommended to use a CO2 chamber or an improvised vessel that could generate CO2 during the incubation period at 42 °C. For best cell growth results on incubated plates, 72 h is recommended for optimum results18.
There are, however, no limitations of this standard procedure, as strict adherence to the steps will ensure viable and pure LAB cells for fermentative procedures. The significance of this standard protocol is based on its proactive approach to ensuring probiotic cell viability, especially for fermentative cultures. It serves as a quality control mechanism that seeks to eliminate contamination and ultimately promote strong and highly viable cultures that could produce desirable yields of fermentation under standard conditions. In reference to existing or conventional methods, this standard protocol eliminates the chance of LAB cell viability challenges (due to cold shock, and poor growth medium) it also seeks to ensure the first-time-right concept of having pure and viable probiotic cultures, saves time, and promotes productivity when working with LAB cultures.
The future applications of this technique are enormous, as this protocol could be used in both research laboratories, dairy and food industries, bioprocessing, fermentation, and biotechnology institutes. The added advantage of this protocol is based on its simplistic nature and thus does not require sophisticated use of laboratory equipment. As such, this protocol could be used for demonstration or pedagogical learning in high schools that have a basic laboratory environment to encourage and build the interest of students in Science, Technology, Engineering, and Mathematics (STEM) related fields.
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Disclosures
The authors have nothing to disclose.
Acknowledgments
This publication was made possible by grant number NC.X-267-5-12-170-1 from the National Institute of Food and Agriculture (NIFA) and in part by NIZO Food Research BV, The Netherlands, Jarrow Formulas, USA, and the Department of Family and Consumer Sciences and the Agriculture Research Station at North Carolina Agriculture and Technical State University (Greensboro, NC, USA 27411). This work was also supported, in part, by 1890 Capacity Building Program grant no. (2020-38821-31113/project accession no. 021765). This work was also partially supported by the Bulgarian Ministry of Education and Science under the National Research Programme ‘Healthy Foods for a Strong Bio-Economy and Quality of Life’ approved by DCM # 577 / 17.08.2018.
Materials
| Name | Company | Catalog Number | Comments |
| Aniline Blue | Thermo Scientific | R21526 | 25 g |
| Beef extract | Research Products International | 50-197-7509 | 500 g |
| Yeast extract | Fisher Scientific | BP1422-500 | 500 g |
| Calcium Chloride dihydrate | Fisher Scientific | C79-500 | 500 g |
| Dextrose Anhydrous | Fisher Scientific | BP350500 | 500 g |
| D-Fructose | ACROS Organics | AC161355000 | 500 g |
| Difco agar powder | Difco | DF0812-07-1 | 2 kg |
| TPY agar | Difco | 211921 | 500 g |
| Eppendorf microcentrifuge tube (Snap-Cap Microcentrifuge Safe-Lock) | Fisher Scientific | 05-402-12 | 2 mL |
| Glycerol | Thermo Scientific | PI17904 | 500 mL |
| Infrared CO2 Incubator | Forma Scientific | ||
| Lactobacillus delbrueckii subsp. bulgaricus | American Type Culture Collection (ATCC) | ATCC 11842 | |
| Lactobacillus delbrueckii subsp. bulgaricus | Bulgaria | S9 | |
| Lactobacillus delbrueckii subsp. bulgaricus | Bulgaria | LB6 | |
| Lactobacillus delbrueckii subsp. bulgaricus | Food Microbiology and Biotechnology Laboratory (NCATSU) | DAW | |
| Lactobacillus delbrueckii subsp. bulgaricus | Food Microbiology and Biotechnology Laboratory (NCATSU) | E22 | |
| Limosilactobacillus reuteri | Biogai, Raleigh / Food Microbiology and Biotechnology Laboratory (NCATSU) | RD2 | |
| L-Cysteine hydrochloride monohydrate | Sigma-Aldrich | C6852-25G | 25 g |
| Maltose monohydrate | Fisher Scientific | M75-100 | 100 g |
| MRS broth | Neogen | 50-201-5691 | 5 kg |
| Peptone No. 3 | Hach | 50-199-6719 | 500 g |
| Potassium phosphate dibasic (K2HPO4) | Research Products International | 50-712-761 | 500 g |
| Sodium acetate trihydrate | Fisher Scientific | S220-1 | 1 kg |
| Sodium chloride | Fisher Scientific | BP358-1 | 1 kg |
| Sodium pyruvate | Fisher Scientific | BP356-100 | 100 g |
| Test Tubes with Rubber-Lined Screw Caps | Fisher Scientific | FB70125150 | 25 x 150 mm |
| Tween 80 | Fisher Scientific | T164-500 | 500 mL |
| Ultra low freezer | So-Low | ||
| Uracil | ACROS Organics | AC157301000 | 100 g |
| UV- visible spectrophotometer | Thermo Fisher Scientific | Evolution 201 | |
| Vortex Genie 2 | Fisher Scientific | ||
| Yeast extract | Fisher Scientific | BP1422-500 | 500 g |
| Ethanol | Fisher Scientific | T08204K7 | 4 L |
| Hydrochloric Acid (6N (Certified), Fisher Chemical) | Fisher Scientific | SA56-500 | 500 mL |
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